PCDHA9 as a candidate gene for amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease. To identify additional genetic factors, we analyzed exome sequences in a large cohort of Chinese ALS patients and found a homozygous variant (p.L700P) in PCDHA9 in three unrelated patients. We generated Pcdhα9 mutant mice harboring either orthologous point mutation or deletion mutation. These mice develop progressive spinal motor loss, muscle atrophy, and structural/functional abnormalities of the neuromuscular junction, leading to paralysis and early lethality. TDP-43 pathology is detected in the spinal motor neurons of aged mutant mice. Mechanistically, we demonstrate that Pcdha9 mutation causes aberrant activation of FAK and PYK2 in aging spinal cord, and dramatically reduced NKA-α1 expression in motor neurons. Our single nucleus multi-omics analysis reveals disturbed signaling involved in cell adhesion, ion transport, synapse organization, and neuronal survival in aged mutant mice. Together, our results present PCDHA9 as a potential ALS gene and provide insights into its pathogenesis.


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Sample size
All raw and processed data from our study have been deposited in recognized public repositories.The raw sequence data from both snRNA-seq and snATAC-seq, as well as the human samples documented within this study, have been deposited in Genome Sequence Archive (Chen et al., 2021) in National Genomics Data Center (CNCB-NGDC Members and Partners, 2022), China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA007917; HRA003114) that are publicly accessible at https://ngdc.cncb.ac.cn/gsa.Additionally, the processed data derived from snRNA-seq and snATAC-seq are conveniently accessible through the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo)under the SuperSeries accession GEO: GSE234783.Furthermore, the mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org)through the iProX partner repository with the dataset identifier PXD042929.The code utilized for our analysis is available in https://github.com/NeuroXplorer-XuLab/PCDHA9-ALS-Candidate-Gene-Functional-Identification.All code and analysis are available on Zenodo.The DOI is 10.5281/zenodo.10561493.
We totally collected 1,315 ALS cases and 430 controls for whole-exome sequencing (WES), ultra-depth targeted panel sequencing and Sanger sequencing studies.We have collected the sex information for all the individuals and performed sexmatching for cases and controls in the whole-exome and targeted sequencing studies.In the WES study, we included 110 male and 44 female ALS cases, and 66 male and 36 female controls.In the panel sequencing study, we included 167 male and 71 female ALS cases, and 144 male and 82 female controls.The sex was statistically matched between cases and controls in both studies.
All the study participants were self-reported Han Chinese.There was no difference for the races of the study participants and we did not need to control for confounding variables.
In the WES study, the age was 52.8±9.6 years in ALS patients and 66.5±10.0 years in the controls.In the targeted panel sequencing study, the age was 54.5±10.9 years in ALS patients and 69.1±7.4 years in the controls.Since ALS is an age-related disease, we selected much older controls to minimize the risk for recruiting potential late-onset ALS patients as controls.S1.All participants were subjected to the clinical neurological assessments, and the patients were all evaluated by the needle EMG study.ALS was diagnosed according to the Gold Coast (GC) criteria for ALS (Shefner et al., 2020) by at least two neurologists.The healthy controls did not have any nervous system or psychiatric diseases.Written informed consent was obtained from all the participants and blood samples were obtained afterwards.

Antibodies
Antibodies used

Validation
were not enough mice available, thus we used all Del mice measured.For other experiment using animal tissues, we used n>3 biology repeats, also in the RNA-seq and ATAC-seq sample, all the sample were used randomization whenever there were age fitted mice, and littermates were used as possible as we could.
No data were excluded from the analyses.
All attempts at replication were successful in 3 times reproduce experimental findings in cell transfection and subsequent WB, IF or IP assays.
For the tissue experiments including WB, IF and IHC, 3 biological repeats were performed, in some assay more than 3 biological repeats were used when there were age fitted mice.All behavior test were reproducible for uncertain times, but collected all the data together for the statistical analysis, because the animals were not always enough amount to do the experiments.Usually, we used n>6 in the WT and Mut mice in the behavior tests including footprint, grip force, swimming and rota-rod test; while for the del mice, we didn't breed enough mice before this strain out of fertility because of the pandemic that leading to close of animal facility, n=4 were usually performed in biological repeats.The specific "n" biological repeats were provided in the figure legends.The RNA-seq and ATAC-seq were not repeated, the sample size were n=3 in biological repeats with same gender, and litter-mates WT and Mut mice were used as possible as we could.
The samples with certain genotypes or cell samples were allocation random.
We were blinded to group allocation during data collection and analysis.All primary antibodies were validated according to the specific signal with right panel and molecular weight before they were used in formal research.Detail of validation from the manufacturers as below: 1) NeuN (Abcam, ab104224, 1030423-1, IF); Suitable for: IHC-P, WB, ICC/IF.Reacts with: Mouse, Rat, Human.The antibody was validated by the company, please refer to the manufacturer's description: https://www.abcam.cn/products/primary-antibodies/neun-antibody-1b7-neuronal-marker-ab104224.html.
All investigations were conducted according to the Declaration of Helsinki, and the study was approved by the Institutional Review Boards of the Ethics Committee of Xuanwu Hospital, Capital Medical University.The study was approved by the Ethics Committees of Xuanwu Hospital of Capital Medical University, West China Hospital of Sichuan University, The First Affiliated Hospital of Sun Yat-sen University and The Chinese PLA General Hospital Sample sizes were selected to ensure sufficient statistical power while minimizing the number of animals used, randomization and blinding was used.For the behavior test in the WT and Mut group, we used n>6 animals for each.However, in the behavior test in Del groups, there nature portfolio | reporting summaryWe require information from authors about some types of materials, experimental systems and methods used in many studies.Here, indicate whether each material, system or method listed is relevant to your study.If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.